The present invention relates to antiviral agents comprising a nitrogen-containing heterocyclic carboxamide derivative or a salt thereof.
Nowadays, antiviral agents are selected and put to use in accordance with the objective viruses. For instance, Acyclovir and Vidarabine are used against herpes viruses; Gancicrovir and Foscarnet are used against cytomegalo virus; and interferon is used against hepatitis viruses.
Influenza virus is a central virus of the cold syndrome, which has attacked human being periodically to cause many deaths amounting to tens millions. Although the number of deaths shows a tendency of decrease in the recent years owing to the improvement in hygienic and nutritive conditions, the prevalence of influenza is repeated every year, and it is apprehended that a new virus may appear to cause a wider prevalence.
For prevention of influenza virus, vaccine is used widely, in addition to which low molecular weight substances such as Amantadine and Ribavirin are also used.
Amantadine is used for prevention and treatment of influenza. Its function mechanism is said to consist in inhibiting the fusion between influenza virus and cell membrane, and it is effective against A-type influenza virus. Its problems are, however, that it is ineffective against B type influenza virus, its resistant virus appears, and it causes side effects such as nerve disturbance. Although Rimantadine which is a derivative of Amantadine has a more improved antiviral activity, the problem of side effect is not overcome by it. Ribavirin which is a guanosine derivative shows a viral RNA polymerase-inhibitory activity and is effective upon A type and B type influenza viruses. Its internal use, however, brings about no sufficient clinical effect.
The present invention provides an antiviral agent exhibiting a preventive effect and a therapeutic effect against various viruses, especially influenza viruses.
The present inventors have conducted researches and studies on compounds showing an antiviral activity against various viruses, especially influenza viruses. As a result, it has been found that pyrazine carboxamide derivatives have an anti-influenza virus activity. The inventors conducted further studies to find that nitrogen-containing heterocyclic carboxamide derivatives represented by the following general formula [1]: 
wherein ring A represents a substituted or unsubstituted pyrazine, pyrimidine, pyridazine or triazine ring; R1 represents O or OH; R2 represents a hydrogen atom, an acyl group or a substituted or unsubstituted carbamoylalkyl or carboxyalkyl group; and the broken line represents a single bond or a double bond; or salts thereof exhibit an excellent antiviral activity against A-, B- and C-type of influenza viruses and other various viruses, these compounds have low cytotoxicity and are useful as an antiviral agents of high safety, as well as that novel N-containing heterocyclic carboxamide derivatives represented by the following general formula [1a]: 
wherein ring Axe2x80x2 represents a pyrazine ring substituted with a halogen atom, a hydroxyl group or an oxide group; R1 represents O or OH; R2 represents a hydrogen atom, an acyl group or a substituted or unsubstituted carbamoylalkyl or carboxyalkyl group; and the broken line represents a single bond or a double bond; or salts thereof exhibit an excellent antiviral activity. Based on these findings, the present invention has been accomplished.
The present invention will be described in detail below.
Unless otherwise indicated, the term xe2x80x9chalogen atomxe2x80x9d used in this specification means a fluorine atom, a chlorine atom, a bromine atom or an iodine atom; the term xe2x80x9calkyl groupxe2x80x9d means a straight or branched chain C1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl and the like; the term xe2x80x9calkenyl groupxe2x80x9d means a straight or branched chain C2-6 alkenyl group such as vinyl, allyl and the like; the term xe2x80x9ccycloalkyl groupxe2x80x9d means a C3-6 cycloalkyl group such as cyclopropyl, cyclopentyl, cyclohexyl and the like; the term xe2x80x9calkoxy groupxe2x80x9d means a straight or branched chain C1-6 alkyl-Oxe2x80x94 group such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy and the like; the term xe2x80x9ccycloalkyloxy groupxe2x80x9d means a C3-6 cycloalkyl-Oxe2x80x94 group such as cyclopropyloxy, cyclopentyloxy, cyclohexyloxy and the like; the term xe2x80x9calkylthio groupxe2x80x9d means a straight or branched chain C1-6 alkyl-Sxe2x80x94 group such as methylthio, ethylthio, n-propylthio, isopropylthio, n-butylthio, isobutylthio, sec-butylthio, tert-butylthio, pentylthio and the like; the term xe2x80x9calkylamino groupxe2x80x9d means an amino group substituted with one or more straight or branched chain C1-6 alkyl groups such as methylamino, ethylamino, propylamino, butylamino, pentylamino, hexylamino, dimethylamino, diethylamino, methylethylamino, dipropylamino, dibutylamino, dipentylamino and the like; the term xe2x80x9ccycloalkylamino groupxe2x80x9d means a C3-6 cycloalkyl-NHxe2x80x94 group such as cyclopropylamino, cyclopentylamino, cyclohexylamino and the like; the term xe2x80x9chalogenoalkyl groupxe2x80x9d means a halogen-substituted C1-6 alkyl group such as trifluoromethyl, trichloro-methyl, chloromethyl and the like; the term xe2x80x9caryl groupxe2x80x9d means a phenyl group, a naphthyl group and the like; the term xe2x80x9caryloxy groupxe2x80x9d means an aryl-Oxe2x80x94 group such as phenyloxy, naphthyloxy and the like; the term xe2x80x9carylthio groupxe2x80x9d means an aryl-Sxe2x80x94 group such as phenylthio, naphthylthio and the like; the term xe2x80x9carylamino groupxe2x80x9d means an aryl-NHxe2x80x94 group such as phenylamino, naphthylamino and the like; the term xe2x80x9cacyl groupxe2x80x9d means a C2-5 alkanoyl group such as formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl and the like and an aroyl group such as benzoyl, naphthoyl and the like; the term xe2x80x9calkoxycarbonyl groupxe2x80x9d means a straight or branched chain C1-6 alkoxycarbonyl group such as methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, isopropoxycarbonyl, n-butoxycarbonyl, isobutoxycarbonyl, sec-butoxycarbonyl, tert-butoxycarbonyl, pentyloxycarbonyl and the like; the term xe2x80x9calkylcarbamoyl groupxe2x80x9d means a carbamoyl group substituted with one or more straight or branched chain C1-6 alkyl groups such as methylcarbamoyl, dimethylcarbamoyl and the like; the term xe2x80x9ccarbamoylalkyl groupxe2x80x9d means a straight or branched chain C1-6 alkyl group substituted with a carbamoyl group such as carbamoylmethyl, carbamoylethyl, carbamoylisopropyl and the like; the term xe2x80x9ccarboxyalkyl groupxe2x80x9d means a straight or branched chain C1-6 alkyl group substituted with a carboxyl group such as carboxymethyl, carboxylethyl, carboxyisopropyl and the like; the term xe2x80x9cheterocyclic groupxe2x80x9d means a 4-, 5- or 6-membered ring or a fused ring thereof having, as the hetero atoms constituting said ring, at least one heteroatoms selected from the group consisting of oxygen atom, nitrogen atom and sulfur atom, such as oxetanyl, thietanyl, azetidinyl, furyl, pyrrolyl, thienyl, oxazolyl, isoxazolyl, imidazolyl, thiazolyl, isothiazolyl, pyrrolidinyl, benzofuranyl, benzothiazolyl, pyridyl, quinolyl, pyrimidinyl and morpholinyl; and the term xe2x80x9coxide groupxe2x80x9d means an oxygen atom linked to a nitrogen atom in a ring. The term xe2x80x9clowerxe2x80x9d means that the number of carbon atoms is 1 to 6.
The protecting group for carboxyl group includes any groups which can conventionally be used as a protecting group for carboxyl group. The examples thereof include alkyl groups such as methyl, ethyl, n-propyl, isopropyl, 1,1-dimethylpropyl, n-butyl, tert-butyl and the like; aryl groups such as phenyl, naphthyl and the like; aralkyl groups such as benzyl, diphenylmethyl, trityl, p-nitrobenzyl, p-methoxybenzyl, bis(p-methoxyphenyl)methyl and the like; acylalkyl groups such as acetylmethyl, benzoylmethyl, p-nitro-benzoylmethyl, p-bromobenzoylmethyl, p-methanesulfonylbenzoylmethyl and the like; oxygen-containing heterocyclic groups such as 2-tetrahydropyranyl, 2-tetrahydrofuranyl and the like; halogeno-alkyl groups such as 2,2,2-trichloroethyl and the like; alkylsilylalkyl groups such as 2-(trimethylsilyl)ethyl and the like; acyloxyalkyl groups such as acetoxymethyl, propionyloxymethyl, pivaloyloxymethyl and the like; nitrogen-containing heterocyclic alkyl groups such as phthalimidomethyl, succinimidomethyl and the like; cycloalkyl groups such as cyclohexyl and the like; alkoxyalkyl groups such as methoxymethyl, ethoxymethyl, 2-(trimethylsilyl)ethoxymethyl and the like; aralkoxyalkyl groups such as benzyloxymethyl and the like; alkylthioalkyl groups such as methylthiomethyl, 2-methylthioethyl and the like; arylthioalkyl groups such as phenylthiomethyl and the like; alkenyl groups such as 1,1-dimethyl-2-propenyl, 3-methyl-3-butenyl, allyl and the like; substituted silyl groups such as trimethylsilyl, triethylsilyl, triisopropylsilyl, diethylisopropylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, diphenylmethylsilyl, tert-butylmethoxyphenylsilyl and the like; etc.
In general formula [1], ring A represents a pyrazine ring, a pyrimidine ring, a pyridazine ring or a triazine ring. More specifically, ring A represents any of the following structures: 
In the structures above, the mark *1 expresses the position of substitution with R1, and the mark *2 expresses the position of substitution with xe2x80x94C(xe2x95x90O)NHR2.
The carbamoylalkyl or carboxyalkyl group represented by R2 may be substituted with at least one substituent selected from the group consisting of halogen atoms; alkyl groups unsubstituted or substituted with hydroxyl, alkoxy, alkylthio, aryl, amino or alkylamino groups; halogenoalkyl groups; alkenyl groups; cycloalkyl groups; hydroxyl groups; alkoxy groups; cycloalkyloxy groups; alkoxycarbonyl groups; mercapto groups; alkylthio groups unsubstituted or substituted with one or more aryl groups; aryl groups; aryloxy groups; arylthio groups; arylamino groups; cyano groups; nitro groups; amino groups unsubstituted or substituted with one or more acyl groups; alkylamino groups; cycloalkylamino groups; acyl groups; hydrazino groups; carboxyl groups; carbamoyl groups; thiocarbamoyl groups; alkylcarbamoyl groups and heterocyclic groups.
Of ring A represented by general formula [1], preferred are pyrazine ring, pyrimidine ring and triazine ring, and more preferred is pyrazine ring. The substituent on ring A includes groups selected from the group consisting of halogen atoms; alkyl groups unsubstituted or substituted with one or more hydroxyl, alkoxy, alkylthio, aryl, amino or alkylamino groups; halogenoalkyl groups; alkenyl groups; cycloalkyl groups; hydroxyl groups; alkoxy groups; cycloalkyloxy groups; alkoxycarbonyl groups; mercapto groups; alkylthio groups unsubstituted or substituted with one or more aryl groups; aryl groups; aryloxy groups; arylthio groups; arylamino groups; cyano groups; nitro groups; amino groups unsubstituted or substituted with one or more acyl groups; alkylamino groups; cycloalkylamino groups; acyl groups; hydrazino groups; carboxyl groups; carbamoyl groups; thiocarbamoyl groups, alkylcarbamoyl groups and heterocyclic groups. Ring A can have one or more of the above-mentioned substituents. Further, the substituent on ring A is preferably linked to carbon atom of the ring.
The salt of the compound of general formula [1] includes any usually known salts formed at the site of basic groups such as amino group and salts formed at the site of acidic group such as hydroxyl and carboxyl groups. The salts formed at the site of basic group include salts of mineral acids such as hydrochloric acid, hydrobromic acid, sulfuric acid and the like; salts of organic carboxylic acids such as tartaric acid, formic acid, citric acid, trichloroacetic acid, trifluoroacetic acid and the like, and salts of sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, mesitylenesulfonic acid, naphthalenesulfonic acid and the like. The salts formed at the site of acidic group include salts of alkali metals such as sodium, potassium and the like; salts of alkaline earth metals such as calcium, magnesium and the like; ammonium salts; and salts of nitrogen-containing organic bases such as trimethylamine, triethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, diethylamine, dicyclohexylamine, procaine, dibenzylamine, N-benzyl-xcex2-phenethylamine, 1-ephenamine, N,Nxe2x80x2-dibenzylethylenediamine and the like. Among the salts mentioned above, preferred are pharmacologically acceptable ones.
Typical compounds represented by general formula [1] are shown in Tables 1 to 4.
In the tables shown below, the abbreviations have the following meanings:
Me: methyl
Et: ethyl
iPr: isopropyl
tBu: tert-butyl,
Ph: phenyl
Ac: acetyl
Bz: benzoyl
R2a: xe2x80x94CH (COOH) CH2COOH
R2b: xe2x80x94CH (CH3) CONHCH (CH3) COOH
The nitrogen-containing heterocyclic carboxamide derivatives represented by general formula [1] or salts thereof are commercially available or can be produced according to any of known processes or analogous processes or by a combination thereof. As the papers describing the production processes thereof, J. Am. Chem. Soc., 71, 78 (1949); J. Am. Chem. Soc., 78, 242-244 (1956); J. Heterocycl. Chem., 15(4), 665-670 (1978); J. Chem. Soc., 1379 (1955); U.S. Pat. No. 5,597,823; etc. can be referred to.
More specifically, the nitrogen-containing heterocyclic carboxamide derivatives represented by general formula [1] or salts thereof can be produced according to the following Production Processes 1 to 3.
Production Process 1
wherein R1, R2, ring A and broken line are as defined above, and R3 represents a protecting group for carboxyl group.
(1-a)
A compound of general formula [1] can be obtained by reacting a compound of general formula [2] with a compound of general formula [4].
The solvent which can be used in this reaction is not particularly limited, so far as it causes no adverse effect on the reaction. The examples of the solvent include alcohols such as methanol, ethanol, isopropyl alcohol and the like; halogenated hydrocarbons such as dichloromethane, chloroform, dichloroethane and the like; aromatic hydrocarbons such as benzene, toluene, xylene and the like; ethers such as dioxane, tetrahydrofuran, anisole, diethylene glycol diethyl ether, dimethyl cellosolve and the like; nitriles such as acetonitrile and the like; amides such as N,N-dimethylformamide, N,N-dimethylacetamide and the like; and sulfoxides such as dimethyl sulfoxide and the like. These solvents can be used in admixture.
The compound of general formula [4] is used at least in an equimolar amount to the compound of general formula [2], and preferably in an amount of 1.0-5.0 mole per mole of the compound of general formula [2].
This reaction can be carried out usually at 0-100xc2x0 C. and preferably at 20-80xc2x0 C., for a period of 5-24 hours and preferably for 30 minutes to 10 hours.
(1-b)
A compound of general formula [1] can be obtained by subjecting a compound of general formula [3] and a compound of general formula [4] to a dehydrating condensation reaction.
The solvent which can be used in this reaction is not particularly limited, so far as it causes no adverse effect on the reaction. The examples of the solvent include ethers such as dioxane, tetrahydrofuran, anisole, diethylene glycol diethyl ether, dimethyl cellosolve and the like; halogenated hydrocarbons such as dichloromethane, chloroform, dichloroethane and the like; and amides such as N,N-dimethylformamide, N,N-dimethylacetamide and the like. These solvents can be used in admixture.
The compound of general formula [4] can be used at least in an equimolar amount to the compound of general formula [3] and preferably in an amount of 1.0-2.0 mole per mole of the compound of general formula [3].
The dehydrating condensing agent which can be used in this reaction includes, for example, 1,3-dicyclohexyl carbodiimide, N,Nxe2x80x2-carbonyl diimidazole, 1-ethyl-3-(3xe2x80x2-dimethylaminopropyl)carbodiimide and the like.
The dehydrating condensing agent can be used at least in an equimolar amount to the compound of general formula [3] and preferably in an amount of 1.0-2.0 mole per mole of the compound of general formula [3].
This reaction can be carried out usually at 0-100xc2x0 C. and preferably at 20-60xc2x0 C., for a period of 5 minutes to 24 hours and preferably for 30 minutes to 10 hours.
Production Process 2
wherein R1, R2, ring A and broken line are as defined above, and R4 represents a lower alkyl group.
A compound of general formula [1] can be obtained by subjecting a compound of general formula [5] to alkyl-ether scission.
More specifically, in a case where R4 is a methyl group, the reaction can be carried out according to the description of PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, Second edition, JOHN WILEY and SONS, pp. 145-199 (1991) or by an analogous method.
Production Process 3
wherein R1, ring A and broken line are as defined above, and R2a represents an acyl group.
A compound of general formula [1c] can be obtained by subjecting a compound of general formula [1b] to acylation in the presence of an acid-eliminating agent.
The solvent which can be used in this reaction is not particularly limited, so far as it causes no adverse effect on the reaction. The examples of the solvent include ethers such as dioxane, tetrahydrofuran, anisole, diethylene glycol diethyl ether, dimethyl cellosolve and the like; aromatic hydrocarbons such as benzene, toluene, xylene and the like; halogenated hydrocarbons such as dichloromethane, chloroform, dichloroethane and the like; amides such as N,N-dimethylformamide, N,N-dimethylacetamide and the like; water; etc. These solvents can be used in admixture.
The acylating agent can be used at least in an equimolar amount to the compound of general formula [1b] and preferably in an amount of 1.0-2.0 mole per mole of the compound of general formula [1b].
The acid-eliminating agent used in this reaction includes, for example, pyridine, triethyl-amine, sodium hydrogen carbonate and the like.
The acid-eliminating agent can be used at least in an equimolar amount to the compound of general formula [1b] and preferably in an amount of 1.0-2.0 mole per mole of the compound of general formula [1b].
This reaction can be carried out usually at 0-100xc2x0 C. and preferably at 20-60xc2x0 C., for a period of 5 minutes to 24 hours and preferably for 30 minutes to 10 hours.
In the Production Processes 1-3, the compounds of general formula [2], [3], [4], [5] and [1b] can be replaced with their salt, respectively. As the salts, the same ones as mentioned for the compound of general formula [1] can be used.
Some of the compounds of general formulas [2], [3], [4], [5] and [1b] and salts thereof may have various isomers such as optical isomers and position isomers, and solvated products. In such cases, any of these isomers and solvates may be used in the present invention.
The compound of general formula [lc] thus obtained can be converted to salts thereof. The salts include the same ones as mentioned for the compound of general formula [1].
The objective viruses of the antiviral agent comprising the nitrogen-containing heterocyclic carboxamide derivative represented by general formula [1] or salt thereof according to the present invention include A-, B- and C-type influenza viruses, papilloma virus, adeno virus, A type hepatitis virus, B type hepatitis virus, C type hepatitis virus, bovine viral diarrhea virus (surrogate virus for C type hepatitis virus), polio virus, echovirus, Coxsackie virus, entero virus, rhino virus, rota virus, Newcastle disease virus, mumps virus, vesicular stomatitis virus, respiratory syncytial virus, and Japanese encephalitis virus. The antiviral agent of the present invention exhibits an especially high effect against influenza viruses.
By combining the nitrogen-containing heterocyclic carboxamide derivatives of the present invention represented by general formula [1] or salts thereof with conventional known excipients, adjuvants and additives, pharmaceutical preparations such as solutions, suspensions, powders, granules, fine granules, tablets, capsules, syrups, elixirs, spirits, troches, gargles, aerosols, etc. can be obtained. These pharmaceutical preparations can be administered either orally or non-orally, namely by injection, percutaneous administration, intrarectal administration, intranarial administration, etc.
The method of administration, dosage and frequency of administration of the antiviral agent of the present invention can be properly selected depending upon the age, body weight and symptom of the patient. Usually 1 to 10 mg/kg of the nitrogen-containing heterocyclic carboxamide derivative or a salt thereof can be administered to an adult either at once or in several portions.
Next, antiviral activity and cytotoxicity of the nitrogen-containing heterocyclic carboxamide derivatives of the present invention represented by general formula [1] or salts thereof will be explained.
Sample:
A nitrogen-containing heterocyclic carboxamide derivative represented by general formula [1] or a salt thereof was dissolved in dimethyl sulfoxide to prepare a solution having a concentration of 10 mg/mL. At the time of use, the solution was diluted with a culture medium to a predetermined concentration and then put to use.
As the host cell of influenza virus, MDCK cell (canine kidney cell) was used. For the cytotoxicity test, Vero cell (monkey kidney cell) was used.
Culture medium:
In the multiplication of MDCK cell and Vero cell and in the cytotoxicity test using Vero cell, Exe2x80x2-MEM (product of Nissui) to which 10% fetal bovine serum had been added was used.
In the measurement of antiviral activity, Exe2x80x2-MEM (product of Nissui) to which 1% bovine serum albumin had been added was used.
(Anti-influenza Activity)
MDCK cells were seeded to a 6-well plate (product of CORNING) at 5xc3x97105 cells/well, and cultured overnight at 35xc2x0 C. in 5% CO2-air atmosphere. Then, the cultured MDCK cells on the plate was treated with influenza virus A/PR/8/34 strain diluted with a serum-free culture medium at the concentration of 200 PFU/mL, at 0.5 mL/well for one hour to achieve inoculation and adsorption. After completion of the inoculation and adsorption, an Exe2x80x2-MEM culture medium containing 0.6% Agar Noble, 1% bovine serum albumin and 3 xcexcg/mL acetyltrypsin and also containing a test compound at a prescribed concentration was added to the cells. After sufficient coagulation, the plate was turned upside down, and cultured for 3 days. After completion of the culture, the alive cells were dyed with 1% Neutral Red. Then, cells were fixed with 10% formalin. The agar medium was removed therefrom with running water. Thereafter, the number of plaques was counted. The plaque inhibition rate was expressed in percentage calculated in comparison with control containing no test compound.
The results are shown in Table 5, wherein the test compound number are the same as those in Tables 1 to 4.
(Cytotoxic Activity)
A culture medium containing a predetermined concentration of test compound was added to a 96-well plate (product of CORNING) in 100 xcexcl/well. Then, Vero cells were adjusted to the concentration of 2xc3x97104 cells/mL with culture medium. The solution was added to the plate at 100 xcexcL/well, and cultured at 37xc2x0 C. in 5% CO2-air atmosphere for 4 days. When the culture was completed, the number of alive cells was counted according to the XTT method [for example, see CANCER RESEARCH, Vol. 48, Pages 4827-4833 (1988)].
As a result, the 50% cell growth inhibitory concentration (IC50) of 3-hydroxy-2-pyrazinecarboxamide (compound No. 1) was 250 xcexcg/mL or more.
(Anti-Rhino Activity)
The confluent monolayers of HeLa cells in 6-well tissue culture plates were inoculated with 70 PFU/well of rhino virus typeII strain. After 60 min, the inoculum was removed, and the test medium (Exe2x80x2MEM) containing the concentration of 30 xcexcg/ml of compounds, 30 xcexcg/ml DEAE-dextran, 30 mM MgCl2, 3% basal medium Eagle vitamine solution, 2% fatal calf serum and 0.6% agarose were added. HeLa cells inoculated with rhino virus were incubated for 3 days at 33xc2x0 C. under 100% humidity. After incubation period, the test plates were fixed with 3% formaldehyde solution and the overlay was removed. The cell-monolayer was stained with 0.05% methylene blue and the plaque numbers were counted. The inhibition percent were calculated from the number of plaque in the treated wells and that in the control wells.
The results are shown in Table 6, wherein the test compound number are the same as those in Tables 1 to 4.
(Anti-RSV Activity)
The confluent monolayers of Hep-2 cells in 6-well tissue culture plates were inoculated with 70 PFU/well of respiratory syncytial virus (RSV) A-2 strain. After 60 min, the inoculum was removed, and the test medium (Exe2x80x2MEM) containing the concentration of 30 xcexcg/ml of compounds, 0.12% glutamine, 2% fatal calf serum and 1% methyl cellulose were added. Hep-2 cells inoculated with RSV were incubated for 3 days at 35xc2x0 C. under 100% humidity. After incubation period, the test plates were fixed with 3% formaldehyde solution and the overlay was removed. The cell-monolayer was stained with Giemsaxe2x80x2 solution and the plaque numbers were counted. The inhibition percent were calculated from the number of plaque in the treated wells and that in the control wells.
The results are shown in Table 7, wherein the test compound number are the same as those in Tables 1 to 4.
(Anti-BVDV Activity)
The confluent monolayers of MDBK cell in 6-well tissue culture plates were inoculated with 70 PFU/well of bovine viral diarrhea virus (BVDV) NADL strain. After 60 min, the inoculum was removed, and the test medium (Exe2x80x2MEM) containing the concentration of 30 xcexcg/ml of compounds, 5% horse serum and 1% SeaPlaque Agar were added. MDBK cells inoculated with BVDV were incubated for 3 days at 35xc2x0 C. under 100% humidity. After incubation period, the test plates were fixed with 3% formaldehyde solution, the overlay was removed, and the cells were stained with 1% crystal violet.
The inhibition percent were calculated from the number of plaque in the treated wells and that in the control wells.
The results are shown in Table 8, wherein the test compound number are the same as those in Tables 1 to 4.